Salidroside and Tyrosol Exert Anxiolytic Effects by Modulating Brain Endogenous Phenols via 4-Hydroxyphenyllactic Acid as a Key Functional Metabolite.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba is a traditional Tibetan medicine that has long been used in China for its cardioprotective, Qi-tonifying, and mind-calming properties. It is traditionally indicated for conditions such as irritability, restlessness, and agitation. Among its major active constituents are two naturally occurring small-molecule phenols-salidroside (SAL) and its aglycone tyrosol (TYR). While SAL has been reported to exert neuropsychiatric effects including anxiolytic activity, the underlying mechanisms remain incompletely understood, and whether TYR shares similar anxiolytic properties has yet to be clarified. AIM OF THE STUDY: To investigate the anxiolytic effects of salidroside and tyrosol and their relationship with brain endogenous small-molecule phenol homeostasis through a targeted phenolomics approach, to identify key differential metabolites, and to validate the anxiogenic effect of 4-hydroxyphenyllactic acid and its involvement in the circadian entrainment pathway. MATERIALS AND METHODS: A murine model of anxiety was established via chronic restraint stress (CRS). The anxiolytic effects of SAL and TYR were evaluated using the open field test (OFT) and the elevated plus maze (EPM) test. A previously established chemical derivatization-UPLC-QqQ-MS/MS method was employed to quantitatively analyze ESMP levels in mouse plasma and brain tissue, with subsequent multivariate statistical analysis to identify key differential ESMPs. Furthermore, behavioral assessments, histopathological examinations (H&E staining, Nissl staining, immunohistochemistry), and transcriptomic sequencing (RNA-Seq) were performed to investigate the impact of key differential ESMPs on anxiety-like behavior in mice and the potential molecular mechanisms involved. RESULTS: Both SAL (50 mg/kg/d) and TYR (23 mg/kg/d) significantly ameliorated anxiety-like behaviors in CRS mice (P < 0.05). Endogenous phenolomics analysis revealed that SAL and TYR interventions could differentially reverse the aberrant expression of multiple ESMPs in the plasma and brain tissue from anxious mice. No common differential ESMPs were identified in plasma. 4-Hydroxyphenyllactic acid (HPLA) and homovanillic acid (HVA) were identified as the common key differential ESMPs in brain tissue, exhibiting significant elevations in the model group (P < 0.05) that were markedly reversed by drug administration. Exogenous administration of HPLA (15 or 30 mg/kg/d) induced pronounced anxiety-like behaviors in mice, concomitant with neuronal shrinkage and increased c-Fos-positive cells in the hippocampus and hypothalamus. Co-administration of SAL or TYR with HPLA significantly reversed HPLA-induced anxiety-like behaviors, establishing HPLA as a functional target of their anxiolytic action. Transcriptomic analysis demonstrated that HPLA treatment was associated with activation of the circadian entrainment signaling pathway in mouse brain tissue (P < 0.001) and upregulated the expression of key genes within this pathway, including Grin2a, Adcy10, and Camk2a (P < 0.05), along with the core circadian regulator Nr1d1 (P < 0.05). CONCLUSIONS: SAL and TYR exert their anxiolytic effects by modulating the levels of multiple ESMPs, notably HVA and HPLA, in the brain. HPLA may be associated with activation of the circadian entrainment signaling pathway, potentially involving alterations in circadian regulatory mechanisms. This study provides novel perspectives for the treatment of anxiety disorders and the discovery of biomarkers.